Treatment of sterol-containing substrates



Patented Nov. 14, 1944 TREATMENT OF STEROL-CONTAINING SUBSTRATES Lester Yoder, Ames, Iowa, asslgnor to Iowa State College Research Foundation, Ames, Iowa, a

corporation of Iowa No Drawing. Application April 22, I942,

Serial No. 440,115

12 Claims. (Cl. zen-397.2)

This invention relates to the treatment of sterol-containing substrates, and more particularly to the isolation and purification of sterols having a single reactive double bond in the hydrophenanthrene nucleus.

Sterols are Iound'in many diflerent materials, and a particularly common source is vegetable and animal fats and oils. For example, cholesterol is found in such animal sources as wool grease, nerve tissue fat, etc. Isocholesterol, which is a mixture of lanosterol and agnosterol, is also found inwool grease and resembles cholesterol inmany respects. Phytosterol occurs in vegetable oils, such as pine oil, tall oil, comoil, etc. All of these sterols possess only a single reactive double bond in the hydrophenanthrene nucleus.

The difllculties in obtaining these various sterols in pureor moderately pure form lie in the separation of the sterols from the materials with which they are mixed. The processe which have heretofore been known and used to separate the sterols from the materialswith which they are found, such as the various vegetable and animal fats and oils, are extremely complicated and are expensive and difllcult to carry out. Moreover, the yields which have been obtained by such processes are very low, and the processes have not been found to be satisfactory for commercial use. h

The invention contemplateslthe treatment of materials containing sterols to separate the ster- 015 from the other materials and to isolate and purify the sterols. The treatment is applicable to mixture containing sterols having a single reactive double bond in the hydrophenanthrene nucleus. Materials containing such sterols, and particularly mixtures of fats and oils in which these sterols are normally found, will hereafter be referred to as sterol-containing substrates.

Broadly, the treatment of the invention includes the reaction of the sterol in the sterolcontaining substrate with oxalic acid to form the oxalic addition product of the sterol and the subsequent separation of the addition product from the mixture. The oxalic acid addition product may be decomposed readily to form the sterol and the acid, and the substantially pure sterol maybe readily separated.

It is desirable in carrying out the process of the invention to convert the sterol content of the substrate to the form of free alcohols, if the sterol is not already in this form. In many substrates, such as nerve tissue fat and tall oil, the sterols are found in the form of free alcohols.

In other substrates, such as wool fat, for example, a substantial portion of the sterols is found in the form of esters, and a preliminary hydrolysis is desirable to convert the sterols to the form oi. free alcohols.

When a portion of the desired sterols is present in the form of esters, the conversion of these compounds to alcohols can be accomplished by saponification of the substrate by any suitable process. For example, saponiflcation may be accomplished by means of the use of a suitable basic ion, such as sodium or potassium hydroxide. In the saponiflcation step, it is important only that hydrolysis occur, and it is not necessary to use a sufllcient excess of the alkali to neutralize thev fatty acids which are formed in the hydrolysis of the fat. However, in practice, an excess oi alkali is usually used in order to insure complete hydrolysis. and the alkali may then be neutralized and the soaps converted to free fatty acids by the subsequent addition of a suitable acid.

If desired, the saponifled portion of the mixture may be separated from the unsaponified fraction, and the latter fraction containing the sterols may be retained for further treatment. However, in many instances it is desirable to retain the free fatty acids formed in the hydrolysis in the mixture which is to be treated since these fatty acids aid in dissolving the oxalic acid which is to be used in the subsequent treatment of the sterol-containing substrate.

The-sterol-containing substrate, which may be either the unsaponifiable fraction of the fat or the hydrolyzed and acidified mixture, contains the sterols having a single reactive double bond in the hydrophenanthrene nucleus in the form of free alcohols. This substrate is dissolved in a substantially anhydrous solvent. Any suitable organic solvent may be used for this purpose. The organic solvent should be one which will dissolve the sterols and in which the oxalic acid is at least moderately soluble. The solvent should also be one in which the sterol oxalic acid addition product is relatively insoluble, or at most only slightly soluble. The solvent should be substantially free of water in order to avoid the decomposition of the acid addition products which are later formed. Moreover, the solvent which is used should not be one which reacts with the oxalic-acid.

It has been found that alcohols and organic solvents in general, which contain oxygen, are not suitable for the process, particularly if the solvent is a short chain compound in which the oxygen is present in any substantial proportion.

The solvents which have been found to be suitable for the process are hydrocarbons and the halogen derivatives of hydrocarbons. By halogen derivatives are meant compounds in which a halogen, such as chlorine or bromine, has been substituted for the hydrogen in one or more atoms of the molecule. Saturated hydrocarbons and their derivatives are particularly suitable.

Ethylene chloride (symmetrical dichlorethane) has been found to be particularly suitable for use as a solvent. Other solvents which may be used are petroleum ether, benzine, carbon tetrachloride, chloroform, and trichlorethylene, although none of these solvents are nearly as satisfactory as ethylene chloride.

The amount of solvent used is not extremely critical and may vary from somewhat less than is necessary to dissolve the substrate to a large excess over that amount. However, a large exces of solvent tends to lower the yield of sterols.

After the substrate has been dissolved in the solvent, the solution is filtered to remove any insoluble material, the sterolsbeing in solution and remainingin the filtrate.

To the filtered solution of the sterol substrate is added a suitable quantity of oxalic acid. Preferably, the quantity of axolic acid used is an excess over the chemically-equivalent quantity necessary for complete reaction with the sterols. Since a single molecule of oxalic acid normally reacts with two molecules of the sterols, the chemically-equivalent quantity of oxalic acid is one-half of the molar equivalent. The oxalic acid should be substantially anhydrous.

The optimum temperature for the reaction with a particular combination of sterol and solvent with the oxalic acid may be determined easily by one skilled in the art. When ethylene chloride is'used as the solvent, the temperature is preferably in the neighborhood of 60 C. to 70 C. This temperature, which is only slightly below the boiling point of the ethylene chloride, serves to dissolve a maximum quantity of the sterol and the oxalic acid while at the same time the acid addition product formed inthe reaction is substantially stable at this temperature. Moreover, a relatively high temperature of this nature is to be preferred, since at lower temperatures there is a gradually-increasing tendency toward precipitation of other products from the solution of the substrate.

' When the sterol-containing substrate and the oxalic acid are dissolved in the solvent, the sterol and the oxalic acid react to form an oxalic acid addition product in the sterol which is relatively insoluble in the solvent and which is precipitated from the solution. Preferably, the solution is cooled after the ingredients are dissolved in order to encourage the crystallizing of the addition product from the solution. If ethylene chloride is used as the solvent and the mixture is dissolved at a temperature of 60 to 70 C., the mixture may be permitted to cool gradually to room temperature and the oxalic acid addition product, will gradually crystallize and be precipitated from the solution. This crystalline precipitate can be separated readily from the solution by filtration or otherwise.

The oxalic acid addition product may be decomposed to form oxalic acid and the sterol by introducing the crystalline product into the water, and preferably by heating the water. The addition product may also be decomposed by the addition of alcohol to the crystalline product. The free sterol may be filtered or otherwise separated from the water or alcohol solution and the oxalic acid may then be recovered from the filtrate. The solvents from which the oxalic acid addition product has been removed, may be recovered by distillation or in any other suitable manner,

When the sterol substrate contains cholesterol and iso-cholesterol, the process may be utilized to obtain both of these sterols and to simultaneously separate them from each other. In this instance, the sterol-containing substrate is reacted with the oxalic acid in a suitable solvent medium. The temperature of the solution is lowered to induce crystallization, and the oxalic acid addition product, which is crystallized out in the first few hours, is found to be substantially pure cholesterol. Thereafter, in further standing, the iso-cholesterol oxalic acid addition product will crystallize from the solution and should be substantially completely separated therefrom at the end of about two days. The solution may be filtered to remove the cholesterol addition product and then again filtered to remove the iso-cholesterol addition product.

The following are specific examples of the process of the invention:

Example 1. 150 gms. 0f wool,fat were heated to 100 0., and 15 gms. of 76% sodium hydroxide in 15 cc. of water were slowly stirred into the heated fat. After heating and stirring the mixture for at least one hour, 60 cc. of hot water were added to the saponlfied mass. While the mixture was still hot, 10 cc. of 96% sulfuric acid in 30 cc. of water was gradually stirred into the heated mass. Approximately 100 cc. of hot water were then added, with thorough mixing, and the mass was let stand in order that it might stratify, the temperature being 80 C. to C. The aqueous layer was drawn oil, and the oil layer was diluted to approximately 350 cc. with ethylene chloride. The ethylene chloride solution was then distilled to remove traces of water, and the residue again diluted to 350 cc. with ethylene chloride. The solution was permitted to stand over night and was then filtered. The filtrate and washings were then diluted to 450 cc. with ethylene chloride.

A 50 cc. aliquot part containing about 2.1 gms. of cholesterol was heated to 65 C. to 70 C., and approximately 1.2 gms. of powdered anhydrous oxalic acid was introduced into the solution. The solution was permitted to stand over night to permit crystallization of the oxalic acid addition product, and the crystals were washed with a small quantity of ethylene chloride. It was found that approximately 3.3 gms. of the cholesterol oxalic acid addition product was obtained.

Example 2,-The unsaponifiable fat from 200 gms. degras, assaying 22 gms. digitonin precipitable cholesterol, in 500 cc. of ethylene chloride is treated at 60 C. with 6 gms. powdered anhydrous oxalic acid and allowed to cool gradually to room temperature. After standing over night, the needles of crude cholesterol oxalic acid are filtered off and washed with 15 to 20 cc. of the solvent. The filtrate is saturated at 60 with an additional 0.6 gm. of the oxalic acid, cooled and chilled to 5 to 10 C. over night. The fine crystalline product about 11 gms. which separates is filtered oil, and the filtrate distilled to about onethird of the original volume. The residue is saturated at 60 C. with 0.6 gm. additional oxalic acid and left to crystallize at least two days when 2 gms. more crude cholesterol oxalic acid can be filtered off.

The 31 gms. of crude product is purified by recrystallization from ethylene chloride below 60 several hours collected and pressed on a filter.

The cake is mixed with 40 cc. of the solvent at 55, cooled gradually and after several hours specific examples of the process have beengiven,

about 5 ms. of the addition product collected on a filter. The combined filtrates are cooled to 5 to 10 for several hours, and the separated material filtered off. The filtrate, distilled to about 35 gms. and saturated with more of the oxalic acid on seeding and standing two days, separates an additional 0.5 gms. or more of the cholesterol addition product.

Upon heating with an excess of water, the addition product decomposes and practically pure cholesterol, about 4 ms, can be filtered 01! and the filtrate evaporated to recover the oxalic acid.

The above process may also be carried out by using petrolic ether instead of the ethylene chloride.

Example 4.--Phytosterol, 6 gms. in 100 cc. of an ethylene chloride solution of an unsaponifiable oil, was heated to 65 C. and 3 gms. powdered anhydrous oxalic acid stirred into it. The addition product separated in needles and was filtered off. It was digested at 60 with 35 cc. additional ethylene chloride, and after cooling 9 gins. crystalline material filtered off. The product was digested in 100 cc. hot water, filtered, and dried to 5.3 gms. of phytosterol.

In the case of sterol-containing substrates having a relatively high proportion of sterol therein, the process is preferably carried out without removing any fats or fatty acids which may be present in the substrate. This is desirable because the cholesterol is normally soluble in a solvent, such as ethylene chloride, to a greater extent than is the oxalic acid, and the presence of the fats and oils of the substrate permits a greater quantity of oxalic acid to be dissolved. For example, if pure cholesterol is to be reacted with oxalic acid in an ethylene chloride solution, it will require a twoor three-step treatment with the solvent and oxalic acid in order to precipitate all of the cholesterol as the acid addition product. When wool grease, for example, is to be treated to remove the cholesterol therefrom, it is desirable that the wool grease be merely hydrolyzed and the entire hydrolyzed wool grease mixture subjected to the treatment with the solvent and the oxalic acid.

The fatty acids in the mixture aid in dissolving the oxalic acid. Such a mixture containing fats or oils, as well as the sterol, will be termed a fatty substrate while th term "fatty acid substrate" will be applied to mixtures containing large amounts of fatty acids which are used in dissolving the oxalic acid.

The process may be applied to the separation of cholesterol, iso-cholesterol, phytosterol, and other sterols having a single reactive double bond in the hydrophenanthrene nucleus.

Although the process has been described in detail for the purpose of illustration and various it will be apparent that many changes and modiflcations may be made without departing from the spirit and scope of the invention.

I claim:

1. In a process for the treatment of sterolcontaining substrates, the step of mixing a substrate containing a sterol having one reactive double bond in the hydrophenanthrene nucleus with oxalic acid in a solvent of the classconsisting of hydrocarbons and halogen derivatives of hydrocarbons to form an oxalic acid addition product of the sterol.

2. In a process for the treatment of sterolcontaining substrates, the step of mixing a substrate containing a sterol having one reactive double bond in the hydrophenanthrene nucleus with. oxalic acid in a liquid halogen derivative of a saturated hydrocarbon to form an oxalic acid addition product of'the sterol.

3. In a process for the treatment of sterolcontaining substrates, the step of mixing a substrate containing a sterol having one reactive double bond in the hydrophenanthrene nucleus with oxalic acid in ethylene chloride to form an oxalic acid addition product of the sterol.

4. In a process for the treating of sterol-containing substrates, the step of reacting a fatty substrate containing a sterol having one reactive double. bond in the hydrophenanthrene nucleus with oxalic acid in a medium consisting predominantly of a liquid halogen derivative of a saturated hydrocarbon.

5. A process for the treatment of sterol-containing substrates, comprising reacting hydrolyzed wool grease with oxalic acid in ethylene chloride to form an oxalic acid addition product of the sterol in the wool grease, and separating the oxalic acid addition product from the mixture.

6. A process for the" treatment of sterol-containing substrates, comprising dissolving oxalic acid and a substrate containing a sterol having one reactive double bond in the hydrophenanthrene nucleus in a solvent of the class consisting of hydrocarbons and halogen derivatives of hydrocarbons, and lowering the temperature of the solution to precipitate therefrom the oxalic acid addition product of the sterol.

7. A process for the treatment of sterol-containing substrates, comprising dissolving oxalic acid and a substrate containing a sterol having one reactive double bond in the hydrophenanthrene nucleus in ethylene chloride at a temperature slightly below the boiling point ofthe ethylene chloride, and cooling the solution to crystallize therefrom the oxalic acid addition product acid with a substrate containing a sterol having one reactive double bond inthe hydrophenanthrene nucleus in a medium comprising a fatty acid substrate and a solvent for the sterol said solvent being a member of the class consisting of hydrocarbons and halogen derivatives of hy drocarbons, and thereafter lowering the tem- ,perature oi the solution to crystallize therefrom 'the oxalic acid addition product 01' the sterol.

10. A process for the treatment of sterol-containing substrates, comprising reacting hydrolyzer woolgrease with oxalic acid in ethylene chloride at a temperature slightly below the boiling point of the ethylene chloride, and lowering the temperature of the solution to crystallize therefrom the oxalic acid addition product oi. the sterol therein.

11. A process for the treatment of sterol-containing substrates, comprising reacting hydrolyzed wool grease with oxalic acid in ethylene chloride at a temperature slightly below the boiling point of the ethylene chloride, separating the crystallized oxalic acid addition product from the solution, and adding water to the crystallized product to recover the sterol.

12. A process for the treatment of sterol-containing substrates, comprising reacting oxalic acid with a substrate containing cholesterol and isocholesterol in ethylene chloride at a temperature slightly below the boiling point 01' the ethylene chloride, lowering the temperature of the solution to crystallize therefrom the cholesterol oxalic acid addition product, filtering the solution to remove the cholesterol oxalic acid addition product, and permitting the solution to stand for a substantial period or time to precipitate the isocholesterol oxalic acid addition product.

' LESTER YODER. 

